Marina N. Asiedu

IL-6- and NGF-Induced Rapid Control of Protein Synthesis and Nociceptive Plasticity via Convergent Signaling to the eIF4F Complex

Despite the emergence of translational control pathways as mediators of nociceptive sensitization, effector molecules and mechanisms responsible for modulating activity in these pathways in pain conditions are largely unknown. We demonstrate that two major algogens, the cytokine interleukin 6 (IL-6) and the neurotrophin nerve growth factor (NGF), which are intimately linked to nociceptive plasticity across preclinical models and human pain conditions, signal primarily through two distinct pathways to enhance translation in sensory neurons by converging onto the eukaryotic initiation factor (eIF) eIF4F complex. We directly demonstrate that the net result of IL-6 and NGF signaling is an enhancement of eIF4F complex formation and an induction of nascent protein synthesis in primary afferent neurons and their axons. Moreover, IL-6- and NGF-induced mechanical nociceptive plasticity is blocked by inhibitors of general and cap-dependent protein synthesis. These results establish IL-6- and NGF-mediated cap-dependent translation of local proteins as a new model for nociceptive plasticity.

Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain.

Spinal protein kinase M £ underlies the maintenance mechanism of persistent nociceptive sensitization

Sensitization of the pain pathway is believed to promote clinical pain disorders. We hypothesized that the persistence of a sensitized state in the spinal dorsal horn might depend on the activity of protein kinase M ζ (PKMζ), an essential mechanism of late long-term potentiation (LTP). To test this hypothesis, we used intraplantar injections of interleukin-6 (IL-6) in mice to elicit a transient allodynic state that endured ∼3 d. After the resolution of IL-6-induced allodynia, a subsequent intraplantar injection of prostaglandin E2 (PGE2) or intrathecal injection of the metabotropic glutamate receptor 1/5 (mGluR1/5) agonist DHPG (dihydroxyphenylglycol) precipitated allodynia and/or nocifensive responses. Intraplantar injection of IL-6 followed immediately by intrathecal injection of a PKMζ inhibitor prevented the expression of subsequent PGE2-induced allodynia. Inhibitors of protein translation were effective in preventing PGE2-induced allodynia when given immediately after IL-6, but not after the initial allodynia had resolved. In contrast, spinal PKMζ inhibition completely abolished both prolonged allodynia to hindpaw PGE2 and enhanced nocifensive behaviors evoked by intrathecal mGluR1/5 agonist injection after the resolution of IL-6-induced allodynia. Moreover, spinal PKMζ inhibition prevented the enhanced response to subsequent stimuli following resolution of hypersensitivity induced by plantar incision. The present findings demonstrate that the spinal cord encodes an engram for persistent nociceptive sensitization that is analogous to molecular mechanisms of late LTP and suggest that spinally directed PKMζ inhibitors may offer therapeutic benefit for injury-induced pain states.

Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

BackgroundDespite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors.ResultsTo test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE2 injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment.ConclusionsThese results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.

Acetazolamide and midazolam act synergistically to inhibit neuropathic pain.

&NA; Treatment of neuropathic pain is a major clinical challenge that has been met with minimal success. After peripheral nerve injury, a decrease in the expression of the K–Cl cotransporter KCC2, a major neuronal Cl− extruder, leads to pathologic alterations in GABAA and glycine receptor function in the spinal cord. The down‐regulation of KCC2 is expected to cause a reduction in Cl− extrusion capacity in dorsal horn neurons, which, together with the depolarizing efflux of Symbol anions via GABAA channels, would result in a decrease in the efficacy of GABAA‐mediated inhibition. Carbonic anhydrases (CA) facilitate intracellular Symbol generation and hence, we hypothesized that inhibition of CAs would enhance the efficacy of GABAergic inhibition in the context of neuropathic pain. Despite the decrease in KCC2 expression, spinal administration of benzodiazepines has been shown to be anti‐allodynic in neuropathic conditions. Thus, we also hypothesized that spinal inhibition of CAs might enhance the anti‐allodynic effects of spinally administered benzodiazepines. Here, we show that inhibition of spinal CA activity with acetazolamide (ACT) reduces neuropathic allodynia. Moreover, we demonstrate that spinal co‐administration of ACT and midazolam (MZL) act synergistically to reduce neuropathic allodynia after peripheral nerve injury. These findings indicate that the combined use of CA inhibitors and benzodiazepines may be effective in the clinical management of neuropathic pain in humans. Symbol. No caption available. Symbol. No caption available.

BDNF regulates atypical PKC at spinal synapses to initiate and maintain a centralized chronic pain state

BackgroundChronic pain is an important medical problem affecting hundreds of millions of people worldwide. Mechanisms underlying the maintenance of chronic pain states are poorly understood but the elucidation of such mechanisms have the potential to reveal novel therapeutics capable of reversing a chronic pain state. We have recently shown that the maintenance of a chronic pain state is dependent on an atypical PKC, PKMζ, but the mechanisms involved in controlling PKMζ in chronic pain are completely unknown. Here we have tested the hypothesis that brain derived neurotrophic factor (BDNF) regulates PKMζ, and possibly other aPKCs, to maintain a centralized chronic pain state.ResultsWe first demonstrate that although other kinases play a role in the initiation of persistent nociceptive sensitization, they are not involved in the maintenance of this chronic pain state indicating that a ZIP-reversible process is responsible for the maintenance of persistent sensitization. We further show that BDNF plays a critical role in initiating and maintaining persistent nociceptive sensitization and that this occurs via a ZIP-reversible process. Moreover, at spinal synapses, BDNF controls PKMζ and PKCλ nascent synthesis via mTORC1 and BDNF enhances PKMζ phosphorylaton. Finally, we show that BDNF signaling to PKMζ and PKCλ is conserved across CNS synapses demonstrating molecular links between pain and memory mechanisms.ConclusionsHence, BDNF is a key regulator of aPKC synthesis and phosphorylation and an essential mediator of the maintenance of a centralized chronic pain state. These findings point to BDNF regulation of aPKC as a potential therapeutic target for the permanent reversal of a chronic pain state.

mTORC1 inhibition induces pain via IRS-1-dependent feedback activation of ERK.

&NA; Using pharmacological and genetic methods, we show that inhibition of mTORC1 induces sensory neuron hyperexcitability and pain via feedback activation of ERK. &NA; Mammalian target of rapamycin complex 1 (mTORC1) inhibitors are extensively used as immunosuppressants to prevent transplant rejection and in treatment of certain cancers. In patients, chronic treatment with rapamycin or its analogues (rapalogues) has been reported to lead to sensory hypersensitivity and pain conditions via an unknown mechanism. Here, we show that pharmacological or genetic inhibition of mTORC1 activates the extracellular signal‐regulated kinase (ERK) pathway in sensory neurons via suppression of S6K1 to insulin receptor substrate 1 negative feedback loop. As a result, increased ERK activity induces sensory neuron sensitization, mechanical hypersensitivity, and spontaneous pain. The clinically available adenosine monophosphate‐activated protein kinase activator, metformin, which is an antidiabetic drug, prevents rapamycin‐induced ERK activation and the development of mechanical hypersensitivity and spontaneous pain. Taken together, our findings demonstrate that activation of the ERK pathway in sensory neurons as a consequence of mTORC1 inhibition leads to the development of pain. Importantly, this effect is abolished by co‐treatment with metformin, thus providing a potential treatment option for rapalogue‐evoked pain. Our findings highlight the physiological relevance of feedback signaling through mTORC1 inhibition and have important implications for development of pain therapeutics that target the mTOR pathway.

The Protease-activated Receptor-2-specific Agonists 2-Aminothiazol-4-yl-LIGRL-NH2 and 6-Aminonicotinyl-LIGRL-NH2 Stimulate Multiple Signaling Pathways to Induce Physiological Responses in Vitro and in Vivo

Protease-activated receptor-2 (PAR2) is one of four protease-activated G-protein-coupled receptors. PAR2 is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR2 reveals a tethered ligand that activates PAR2 and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca2+ signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR2 (e.g. SLIGRL-NH2 and SLIGKV-NH2) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH2 (2-f-LIGRLO-NH2) underscores the use of peptidomimetic PAR2 ligands as a mechanism to enhance pharmacological action at PAR2, 2-f-LIGRLO-NH2 has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH2 and two recently described pentapeptidomimetic PAR2-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at-LIGRL-NH2) and 6-aminonicotinyl-LIGRL-NH2 (6-an-LIGRL-NH2). All peptidomimetic agonists stimulated PAR2-dependent in vitro physiological responses, MAPK signaling, and Ca2+ signaling with an overall rank order of potency of 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ≫ SLIGRL-NH2. Because PAR2 plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca2+ signaling in nociceptors in vitro, and both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 stimulated PAR2-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR2 in a variety of systems and pathologies.

Modulation of Spinal GABAergic Analgesia by Inhibition of Chloride Extrusion Capacity in Mice

UNLABELLED Spinal gamma-aminobutyric acid receptor type A (GABA(A)) receptor modulation with agonists and allosteric modulators evokes analgesia and antinociception. Changes in K(+)-Cl(-) cotransporter isoform 2 (KCC2) expression or function that occur after peripheral nerve injury can result in an impairment in the Cl(-) extrusion capacity of spinal dorsal horn neurons. This, in turn, alters Cl(-)-mediated hyperpolarization via GABA(A) receptor activation, contributing to allodynia or hypersensitivity associated with nerve injury or inflammation. A gap in knowledge exists concerning how this loss of spinal KCC2 activity differentially impacts the analgesic efficacy or potency of GABA(A) agonists and allosteric modulators. We utilized intrathecal drug administration in the tail flick assay to measure the analgesic effects of general GABA(A) agonists muscimol and Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA), the ∂-subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), and allosteric modulators of the benzodiazepine (midazolam) and neurosteroid (ganaxolone) class, alone or in the presence of K(+)-Cl(-) cotransporter isoform (KCC) blockade. Intrathecal muscimol, ZAPA, THIP midazolam, and ganaxolone all evoked significant analgesia in the tail flick test. Coadministration of either agonists or allosteric modulators with [(dihydroindenyl)oxy] alkanoic acid (DIOA) (a drug that blocks KCC2) had no effect on agonist or allosteric modulator potency. On the other hand, the analgesic efficacy of muscimol and ZAPA and the allosteric modulator ganaxolone were markedly reduced whereas THIP and midazolam were unaffected. Finally, in the spared nerve injury model, midazolam significantly reversed tactile hypersensitivity while ganaxolone had no effect. These results indicate that the KCC2-dependent Cl(-) extrusion capacity differentially regulates the analgesic efficacy of agonists and allosteric modulators at the GABA(A) receptor complex. PERSPECTIVE Our work suggests that drug discovery efforts for the treatment of chronic pain disorders should target benzodiazepine or ∂-subunit-containing sites at the GABA(A) complex.

Development of highly potent protease-activated receptor 2 agonists via synthetic lipid tethering

Protease‐activated receptor‐2 (PAR2) is a G‐protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR2 is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR2 is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR2 agonist (2‐aminothiazol‐4‐yl‐LIGRL‐NH2) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high‐throughput physiological assay, these STL agonists displayed EC50 values as low as 1.47 nM, representing a ~200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR2: EC50 for Ca2+ response as low as 3.95 nM; EC50 for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC50 of 14.4 pmol. STLs failed to elicit responses in PAR2 cells at agonist concentrations of >300‐fold their EC50 values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR2 and represent opportunities for drug development at other protease activated receptors and across GPCRs.—Flynn, A. N., Hoffman, J., Tillu, D. V., Sherwood, C. L., Zhang, Z., Patek, R., Asiedu, M. N. K., Vagner, J., Price, T. J., Boitano, S. Development of highly potent protease‐activated receptor 2 agonists via synthetic lipid tethering. FASEB J. 27, 1498–1510 (2013). www.fasebj.org